PROJECT SUMMARY Uveal melanoma (UM) is the most common primary ocular malignancy. In approximately 50% of patients, UM progresses into a metastatic disease, with the liver being the most common site of metastasis. Once this occurs, prognosis is dismal, with a median survival of less than one year. There are very few effective therapies for metastatic UM, in part due to the paucity of knowledge regarding the molecular mechanisms that control the invasion and metastasis of UM cells. A WNT family ligand, WNT5A, has been implicated in the invasion and metastasis of several different cancers, but its role in UM is unknown. One way by which WNT5A promotes these processes is by binding receptor tyrosine kinase-like orphan receptor 2 (ROR2) and stimulating its internalization. This is required to activate downstream targets that control invasion, such as protein kinase C (PKC). The small GTPase ARF6 is required for the invasion of several types of cancer, is activated by WNT5A in cutaneous melanoma, and is known to regulate the trafficking of receptors at the cell surface. Preliminary data suggest that WNT5A expression is elevated and activates ARF6 in some UM cells. The first aim of this research is to determine whether a WNT5A-ROR2-ARF6 axis controls invasion and metastasis of UM. Key methods include Matrigel invasion assays, a murine xenograft model of metastatic UM, and pharmacologic targeting of ARF6 with novel small molecule inhibitors. The second aim is to dissect the mechanism by which ARF6 mediates WNT5A-ROR2 signaling. We will use cellular fractionation assays, immunofluorescence staining and western blotting to study the role of ARF6 in the trafficking and signaling of ROR2. This work has also identified a potential complex containing ROR2 and ?-catenin in the nucleus of UM cells. The other major aim of the proposal is to elucidate the function of nuclear ROR2 and determine whether it alters ?-catenin transcriptional activity to promote invasion and metastasis of UM. This will be accomplished using ROR2 knockout UM cell lines and ectopic expression of ROR2 nuclear localization mutants. mRNA-Seq data from these cells will be analyzed to reveal global expression changes and specific alterations in the ?- catenin transcriptional signature. Overall, we expect the proposed project to reveal a new pathway that could be targeted to combat metastasis of uveal melanoma. This research also has the potential to uncover a critical function of nuclear ROR2 that is likely conserved in other cancers and developmental processes.